1. Field of the Invention
This invention relates to assay methods, and reagent means for use therein, for the determination of one of the coenzymes adenosine triphosphate (ATP) or flavin mononucleotide (FMN) in a liquid medium. In particular, this invention relates to an enzymatic assay for ATP or FMN based on the use of a flavin adenine dinucleotide (FAD) synthetase-active preparation to produce FAD. FAD indicator systems are used to generate a detectable, usually colorimetric, response. FAD synthetase is formally designated as ATP:FMN Adenylyl Transferase (EC 2.7.7.2) by the International Committee of Enzyme Nomenclature.
2. Description of the Prior Art
The production of ATP is a common goal of both anaerobic and aerobic metabolitic activity. Determination of ATP is a direct indication of life, and is a useful index of the presence of microbial biomass and microbiological activity. However, the concentration of ATP in biological fluids is often only 10.sup.-9 moles/liter (nanomolar) to 10.sup.-12 moles/liter (picomolar) or even less, so that detection requires highly sensitive ATP assays.
The enzymatic determination of adenosine phosphates by spectrophotometric methods involving pyridine nucleotides has come into general use because of its simplicity compared with paper or column chromatography methods.
A well known ultraviolet photometric method for the determination of ATP involves the following sequence of reactions [Jaworek, D., et al., in "Methods of Enzymatic Analysis", Section D, 2nd English Ed., H. U. Bergmeyer Ed., Academic Press, New York 2097, (1974)]: ##STR1## where the decrease in absorbance of reduced nicotinamide adenine dinucleotide (NADH) is monitored at 340 nanometers (nm). The NADH disappearance can also be monitored by its native fluorescence which increases the assay sensitivity up to micromolar (10.sup.-6 moles/liter) levels of ATP. However, the method suffers from lack of specificity as other nucleoside triphosphates, including guanosine triphosphate, inosine triphosphate, and uridine triphosphate, can be measured as ATP.
The hexokinase/glucose-6-phosphate dehydrogenase method [Lamprecht, W., et al., in "Methods of Enzymatic Analysis" Section D, 2nd English Ed., H. U. Bergmeyer Ed., Academic Press, New York 2101, (1974)] has also proven valuable in the enzymatic determination of ATP. Highly purified enzymes are mandatory to obtain good specificity with this method.
Another interesting approach involves amplifying sensitivity through a reaction mechanism that casts the analyte itself into a cycling role so that a stoichiometric amount of product is formed and accumulated every time the analyte is cycled [Campbell, J., et al., Bioch. et Biophy. Acta, 397, 101-109 (1975)]. For example, the ATP can be determined by cycling between two enzyme catalyzed reactions to yield a product, e.g., (pyruvate kinase and hexokinase to glucose-6-phosphate), which product is determined by a third enzymatic reaction:
(a) cycling reaction: ##STR2##
(b) monitoring reaction: ##STR3## However, the manipulations required are too cumbersome for routine use.
The concentration of adenosine phosphates in biological fluids can also be determined by bioluminescent methods. In particular ATP in biological fluids can be measured by monitoring the emitted light produced by the following reactions: ##STR4## Such bioluminescent methods are presently preferred as their higher sensitivity allows for an ATP assay in the nanomolar to picomolar range. While an improvement over conventional techniques, emitted light measurement is limited in routine use both because dedicated instrumentation is required for reading and because of the lack of automated instrumentation for sample processing. Therefore, there is a need for improved methods for determining ATP.
Hornby, et al., U.S. Pat. No. 4,238,565, describe the use of an organic prosthetic group, such as FAD, which will combine with an apoenzyme (both inactive components) to form a holoenzyme (active), as a label in a specific binding assay.